Cross-linked layer-by-layer (LbL) assemblies with a precisely tuned surface ζ-potential were fabricated to control the adsorption of proteins, mammalian cells, and bacteria for different biomedical applications. Two weak polyions including a synthesized polyanion and polyethylenimine were assembled under controlled conditions and cross-linked to prepare three robust LbL films as model surfaces with similar roughness and water affinity but displaying negative, zero, and positive net charges at the physiological pH (7.4). These surfaces were tested for their abilities to adsorb proteins, including bovine serum albumin (BSA) and lysozyme (LYZ). In the adsorption tests, the LbL films bind more proteins with opposite charges but less of those with like charges, indicating that electrostatic interactions play a major role in protein adsorption. However, LYZ showed higher nonspecific adsorption than BSA, because of the specific behavior of LYZ molecules, such as stacked multilayer formation during adsorption. To exclude such stacking effects from experiments, protein molecules were covalently immobilized on AFM colloidal probes to measure the adhesion forces against the model surfaces utilizing direct protein molecule-surface contacts. The results confirmed the dominating role of electrostatic forces in protein adhesion. In fibroblast cell and bacteria adhesion tests, similar trends (high adhesion on positively charged surfaces, but much lower on neutral and negatively charged surfaces) were observed because the fibroblast cell and bacterial surfaces studied possess negative potentials. The cross-linked LbL films with improved stability and engineered surface charge described in this study provide an excellent platform to control the behavior of different charged objects and can be utilized in practical biomedical applications.